Generation and characterization of a zebrafish knockout model of abcb4, a homolog of the human multidrug efflux transporter P-glycoprotein

The ATP-binding cassette subfamily B member 1 (ABCB1), encoding a multidrug transporter referred to as P-glycoprotein (Pgp), plays a critical role in the efflux of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. Therefore, developing high throughput animal models to screen for Pgp function and bioavailability of substrates and inhibitors is paramount. Here, we generated and validated a zebrafish knockout line of abcb4, a human Pgp transporter homolog. CRISPR/Cas9 genome editing technology was deployed to generate a frameshift mutation in exon 4 of zebrafish abcb4. The zebrafish abcb4 homozygous mutant exhibited elevated accumulation of fluorescent rhodamine 123, a substrate of human Pgp, in the intestine and brain area of embryos. Moreover, abcb4 knockout embryos were sensitized toward toxic compounds such as doxorubicin and vinblastine compared to the WT zebrafish. Immuno-staining for zebrafish Abcb4 colocalized in the endothelial brain cells of adult zebrafish. Transcriptome profiling using Gene Set Enrichment Analysis (GSEA) uncovered that the ‘cell cycle process,’ ‘mitotic cell cycles,’ and ‘microtubule-based process’ were significantly downregulated in the abcb4 knockout brain with age. This study establishes and validates the abcb4 knockout zebrafish as an animal model to study Pgp function in vivo. Unexpectedly it reveals a potentially novel role for zebrafish abcb4 in age-related changes in the brain. The zebrafish lines generated here will provide a platform to aid in the discovery of modulators of Pgp function as well as the characterization of human mutants thereof.


Introduction
ATP-binding cassette (ABC) transporters are membrane-embedded proteins that actively expend the energy of ATP to e ux substrates to the extracellular space [1,2]. An essential ABC transporter in humans, P-glycoprotein (Pgp), which is encoded by the gene ABCB1, has been shown to play a crucial role in host detoxi cation of xenobiotic substances [2,3], leading to multidrug resistance in tumor cells [4]. Speci cally, Pgp is expressed on the apical side of endothelial cells within the blood-brain barrier (BBB) and intestinal epithelium to redirect potential toxins into the bloodstream and gut lumen [5][6][7].
While Pgp plays a signi cant role in protecting these tissues under normal conditions, its overexpression in tumor cells has been implicated in the refractory treatment of brain malignancies and metastases by chemotherapeutical agents, and in the oral bioavailability of drugs [8].
The direct role of Pgp in drug resistance in conjunction with other ABC transporters has been previously investigated through a mouse model [8][9][10][11]. Abcb1a and Abcb1b (the mouse homologs of human ABCB1) knockout mice displayed higher brain penetration of the Pgp substrate ivermectin, causing severe neurotoxicity and death. [11]. Similarly, these mouse models highlighted the function of Pgp in e ux activities after systemic exposure to substrates [9]. In vitro studies using tissues from Abcb1a knockout mice reported that Pgp modulates drug permeability in the intestinal epithelium. [12]. Thus, mouse models have provided valuable information regarding the function of Pgp. However, they are expensive to maintain and unsuitable for high-throughput screening or non-invasive imaging.
The zebra sh provides distinct advantages as a model for studying the role of ABC transporters such as Pgp. Zebra sh has a structurally similar endothelial membrane system to higher vertebrates, including humans, in the BBB and the intestinal tract [13][14][15]. Although synteny analysis found that zebra sh has two Pgp orthologs, abcb4, and abcb5 [16], high-throughput screening of human Pgp substrates characterized Abcb4 as functionally phenocopied to human Pgp [17]. It has been reported that the C219 antibody that recognizes human Pgp cross-reacts with zebra sh Abcb4 and Abcb5 [15]. Therefore, coupling antibody staining with RNAscope techniques was required to observe Abcb4 localization in zebra sh [17]. However, the precise characterization of Abcb4 protein expression in zebra sh using immunohistochemistry was still not feasible due to the lack of antibodies speci c to zebra sh Abcb4.
Here, we report the generation of an abcb4 knockout zebra sh model via CRISPR/Cas9 genome editing technology. These lines exhibited a higher accumulation of rhodamine 123 in the gut epithelium.
Additionally, abcb4 knockout embryos show increased susceptibility in response to human Pgp substrates such as vinblastine and doxorubicin. Taking advantage of the cross-reactivity of the human Pgp antibody F4 with zebra sh Abcb4, we demonstrate that zebra sh Abcb4 is localized in various barrier sites such as brain vasculature, intestinal epithelium, and kidney tubules and ducts. Indeed, elevated rhodamine 123 intensity in the brain area of abcb4 knockout embryos after intravascular injection suggests that Abcb4 functions as an e ux pump at the BBB. Transcriptome pro ling was performed to investigate the function of abcb4 in the brain, revealing signi cantly downregulated cell cycle-related pathways. Taken together, our ndings established the abcb4 mutant zebra sh as an effective model for Pgp studies in vivo. These lines will provide a platform to investigate potential inhibitors of Pgp and for functional characterization of human mutants via transgenic expression.

Results
Generation and validation of a knockout model of the zebra sh homolog of the human ABCB1 Two orthologs of human ABCB1, abcb4 and abcb5, are found in the zebra sh, of which abcb4 is functionally similar to human ABCB1 [16,17]. To elucidate the in vivo roles of zebra sh abcb4 in more details, we generated an abcb4-mutated zebra sh line using CRISPR/Cas9 genome editing technology. Guide RNA (gRNA) targeting exon 4 of the genomic sequence of abcb4 produced a frameshift mutation in the abcb4 gene leading to a nonsense codon and premature translation termination. Speci cally, the mutant allele with a two-nucleotide deletion in exon 4 was predicted to generate truncated Abcb4 proteins of 85 amino acids (Fig. 1a). The abcb4 transcripts were reduced by 80% in the homozygous knockout embryos, suggesting that the aberrant mRNAs were degraded via nonsense-mediated decay (NMD) (Fig. 1b). In adult zebra sh, the abcb4 transcripts were reduced 80% in the heterozygous, and 90% in homozygous brain tissues (Fig. 1c). No evident phenotypes were detected in the mutants at the embryonic stages (data not shown).
Zebra sh abcb4 knockout lines are defective in the e ux of human Pgp substrates.
Having demonstrated the reduction of abcb4 transcripts, we investigated e ux pump activity in the zebra sh abcb4 mutant using the Human Pgp uorescent substrate Rhodamine123 as a proxy for e ux transporter function [18]. For this purpose, WT and abcb4 mutant embryos at 5 days post fertilization (dpf) were incubated in 50 µM of rhodamine 123 for 2 hours, then the level of uptake of rhodamine 123 was examined with uorescence microscopy. We found higher accumulation of rhodamine 123 in the mid-intestines of the abcb4 mutant embryo compared to the WT (Fig. 2a). Moreover, WT embryos showed rhodamine 123 uorescence primarily in the intestinal lumen, whereas abcb4 mutant embryos displayed the uorescence in the gut epithelium. To quantitively compare rhodamine123 accumulation in the midintestine between WT and abcb4 mutant, the intensity of its uorescence was measured and analyzed by Fiji software [19]. The results con rmed that rhodamine123 was signi cantly higher in the intestine area of the abcb4 mutant (Fig. 2b).
Having con rmed the reduced e ux of rhodamine123 in abcb4 knockout embryos, we performed an embryo cytotoxicity assay [21] with other known substrates of human Pgp, vinblastine and doxorubicin [22], to determine to what extent the chemical resistance of the zebra sh embryo is associated with abcb4 transporter activity. In this assay, embryos were exposed to the compounds 6-48 hours post fertilization (hpf), then developmental defects of embryos, including vertebral malformation and growth retardation, were determined ( Fig. 2c and e). We found that developmental abnormalities were highly elevated in abcb4 mutant embryos in the presence of 2 µM vinblastine compared to WT embryos (Fig. 2d). Toxicity of doxorubicin at 100 µM appears to be low; we did not observe severe developmental malformations in either WT or abcb4 mutant groups. Indeed, substantially higher concentrations of doxorubicin are required to induce lethal effects [23]. However, abcb4 de cient embryos displayed greater growth retardation with doxorubicin treatment (Fig. 2f). The overlapping substrate speci city strongly supports Abcb4 as the functional zebra sh homolog to human Pgp, suggesting our abcb4 knockout zebra sh is a tractable model for screening Pgp substrates.
Organ-speci c expression patterns of Abcb4 in adult zebra sh.
In light of the tissue-speci c expression of zebra sh Abcb4, we sought to localize the expression pattern of Abcb4 to determine whether it could be utilized as a model system of the BBB or for oral drug bioavailability screening. It has been reported that the human Pgp C219 antibody cross-reacts with both zebra sh Abcb4 and Abcb5 [13]. Therefore, to identify antibodies that exhibit cross-reactivity with zebra sh Abcb4 proteins, commercial human Pgp antibodies were screened using immunohistochemistry analysis of adult WT and abcb4 knockout brain tissues. The human Pgp antibody F4 showed zebra sh Abcb4-speci c immunoreactivity in the WT adult zebra sh brain but not in abcb4 knockout tissues ( Fig. 3a and b). Higher magni cation images indicated that the pattern of F4 positive staining in WT zebra sh likely corresponds to the structure of brain vasculature (Fig. 3a).
To con rm zebra sh Abcb4 expression in the brain vessels, we performed immunostaining of the F4 antibody in the k1:GFP transgenic line, which expresses GFP in blood vessel endothelial cells. Our staining showed that expression of zebra sh Abcb4 (red) colocalized with k1:GFP positive endothelial cells (green) throughout the CNS (Fig. 3c). Staining with the F4 antibody of the whole sh ( Fig. 3d) revealed positive F4 signal (red) in the renal tubules of the kidney (Fig. 3e) and in the intestinal epithelium (Fig. 3f) as well as the brain (Fig. 3f). This expression pattern is similar to that of human Pgp [24].
Probing the function of Abcb4 at the blood-brain barrier of zebra sh.
Based on the observation that zebra sh Abcb4 is expressed explicitly in brain vasculature (Fig. 3c), we examined the e ux activity of Abcb4 at the BBB in zebra sh. For this purpose, we performed intracardiac injection of rhodamine123 at 3 dpf embryos of Tg[ k1:EGFP] and Tg[ k1:EGFP];abcb4 −/− and imaged live sh after 0.5 hours of circulation (Fig. S1). The parenchymal intensity of rhodamine123 dye was elevated ~ 5-fold in both groups compared to non-injected groups, and there was no signi cant difference between Tg[ k1:EGFP] and Tg[ k1:EGFP];abcb4 −/− (Fig. S1). This result indicates that the BBB of zebra sh embryo at 3 dpf is permeable, which agrees with previous studies that the BBB is functionally immature at 3 dpf [13,25]. Interestingly, images of injected embryos after 1.5 hours of circulation showed that the level of rhodamine123 remaining in the parenchyma of Tg[ k1:EGFP];abcb4 −/− was signi cantly higher than that of Tg[ k1:EGFP] (Fig. 4b and c). The result suggests that Abcb4 functions as an e ux pump of rhodamine123 in the brain of 3 dpf zebra sh embryos. We note that this nding is in contrast with a previous study that reported the lack of rhodamine123 transport in the brain of 3 dpf zebra sh larvae [13].
Age-related changes in the zebra sh brain transcriptome due to loss of abcb4 function.
Age-associated decline in Pgp function could facilitate the accumulation of toxic substances in the brain, thus increasing the risk of neurodegenerative pathology with aging [10]. To gain insight into the function of Abcb4 in the brain, especially how age-related xenobiotic accumulation alters global molecular regulation, we performed brain transcriptome analysis of WT and abcb4 knockout at two different age groups. RNA-seq analysis identi ed Differentially Expressed (DE) genes with FDR cut-off ≤ 0.05 between WT and abcb4 knockout brain tissues at 2 and 30 months (Fig. 5). At 2 months, there were only 22 DE genes between WT and abcb4 de cient brains. However, at 30 months, the number of DE genes in brain tissue between WT and abcb4 knockout sh increased to 294, suggesting that the loss of abcb4 on the brain transcriptome is aggravated with age (Fig. 5a, see Additional le1 for DE gene list).
To derive a global understanding of age-related molecular signatures in WT and abcb4 de cient brains, the DE genes between groups were used for further Gene set enrichment analysis (GSEA) using the WEBbased GEne SeT AnaLysis (WebGestalt) Toolkit (see Additional le 2 for detailed genes lists of GSEA) [26]. In WT, positively enriched categories between 2 and 30 months included 'response to external stimulus,' 'regulation of signaling receptor activity, 'cellular response to organic substances,' and 'cytokine response' (Fig. 5b, e). Speci cally, mRNA level of chemokine ligands such as ccl34b.4 and ccl36.1 were upregulated in WT brain with age. However, these positively regulated pathways in WT with aging were not detected in abcb4 de cient brain. Conversely, genes associated with oxidative stress are upregulated in the abcb4 knockout brain but not in WT (Fig. 5c, e). In the age comparisons between 2 and 30 months of WT and abcb4 knockout brain, both showed 'mitotic cell cycle,' 'cell cycle process,' and 'microtubulebased process' as negatively enriched categories (Fig. 5b, c), which are pathways associated with cell division. Moreover, the three pathways are more signi cantly down-regulated in the abcb4 knockout-aged brain than in WT. Thus, more genes involved in the cell division-related pathways are negatively regulated in the abcb4-depleted brain with age (Fig. 5e).

Discussion
The study reported here takes advantage of the power of zebra sh as a model organism to generate the rst knockout model of abcb4, a functional homolog of human Pgp. In zebra sh, Abcb4 and Abcb5 are both associated with e ux transport activities. However, zebra sh Abcb4 protein has a highly overlapping substrate speci city pro le with human Pgp [17]. In addition, previous studies based on morpholino knockdown found that zebra sh Abcb4 transports several uorescent Pgp substrates in embryos [16]. Our work expands on these previous studies by establishing an abcb4 knockout animal model. We showed that abcb4 mutant embryos exhibited a higher accumulation of rhodamine123 in the gastrointestinal tract (Fig. 2a). Interestingly we note that rhodamine123 accumulation in the mid-intestine of abcb4 knockout embryos overlaps with lysosome-rich enterocytes (LREs) that internalize dietary protein via receptor-mediated and uid-phase endocytosis for intracellular digestion and trans-cellular transport [20]. This observation could suggest that zebra sh abcb4 plays a vital function in the lysosomal tra cking of substrates in intestinal cells, although further detailed analysis is needed. In addition to the established role of plasma membrane Pgp, lysosomal Pgp has also been shown to transport cytotoxic agents [27,28]. Thus, our results suggest a similar role for Abcb4 in the lysosomal membrane of the zebra sh gastrointestinal tract.
We observed high reactivity of the Abcb4-speci c antibody F4 in the gastrointestinal tract and renal kidney tubes ( Fig. 3e and f), suggesting a high level of Abcb4 expression in the zebra sh gut and kidney.
We also noted F4 antibody reactivity in the endothelial cells of the zebra sh brain, indicating that Abcb4 is expressed at the BBB. Moreover, zebra sh Abcb4 functions as an e ux transporter at 3 dpf embryos ( Fig. 4 and Fig. S1). Thus, our ndings suggest that the abcb4 knockout could serve as a powerful zebra sh model, including penetration of drugs at the BBB and pre-clinical examination of oral drug bioavailability and disposition.
A previous human study reported a decrease in Pgp function in the BBB with age [29]. In addition, it has been reported that Pgp de ciency at BBB increases Aβ−deposition in an Alzheimer disease (AD) mouse model [30]. Thus, in mammals, the age-dependent loss of Pgp function may be involved in developing age-related disorders. We re-analyzed a previous zebra sh brain transcriptome and found that the level of abcb4 transcript is not changed with age [31], which agrees with our RNA-seq data. However, the protein level of zebra sh Abcb4 or the transporter activities of Abcb4 may be modulated with age. Examining the changes in the level of Abcb4 protein in old zebra sh is underway to test this possibility.
The pathway analysis of RNA-seq between WT and abcb4 knockout brain tissues at different ages brings to the forefront a critical role of Abcb4 and possibly human Pgp in aging. The observation of a negative correlation between cell cycle-related pathways and aging in the transcriptome of WT zebra sh brain suggests that downregulation of the cell cycle-related pathways is part of normal aging, yet it is potentiated in the abcb4 knockout. Therefore, an important question is whether toxic substances accumulating in the abcb4 depleted zebra sh brain may cause this further downregulation. Interestingly, it has been suggested that senescence-associated signatures are correlated with increasing aneuploidy and genomic instability due to the downregulation of genes involved in the cell cycle and mitosis progression [32][33][34][35]. For example, cenpe, one of the core genes encoding protein involved in spindle assembly and chromosome segregation, is downregulated after the onset of senescence [34,36]. Indeed, the mRNA levels of key players in the cell cycle, including cenpe and aspm, were dramatically reduced in the brain tissue of the abcb4 mutant at 30 months compared to WT (Fig. 5e). If so, loss of abcb4 may play a vital role in inducing an accelerated senescence process in the brain by increasing genome instability, although further experiments are needed to understand the underlying mechanisms.

Materials and methods
Zebra sh maintenance and breeding AB wild-type strain zebra sh (Danio rerio) were used. The embryos were obtained by natural spawning and raised at 28.5°C on a 14:10 h light/dark cycle in egg water 30 mg/L instant ocean in deionized water. Embryos were staged according to their ages (in dpf). All animal procedures were approved by the Vanderbilt University Institutional Animal Care and Use Committee. For abcb4 knockout genotyping, forward primer 5'-CTTGGC TTAATCATGTCGATGGCCA − 3' and reverse primer 5' -TGTCATCTTCTCCCCCAAAG -3' were used for PCR ampli cation. The resulting PCR products were digested with the restriction enzyme NcoI to identify the WT and mutant genotypes.

RNA-Seq
Total RNA from zebra sh brain tissues was isolated using TRIzol Measurement of e ux transporter activity in embryos with rhodamine123 uorescent dye 10 embryos at 5 dpf were placed in one well of a 24-well plate (polystyrene, tissue culture grade) and incubated with 1 ml of 50 µM rhodamine123 (Invitrogen, R302) diluted in 0.3x Danieau water for 2 hours in the dark and rinsed three times with 0.3x Danieau water to remove excess dye. The amount of rhodamine123 accumulated in the gut area of zebra sh embryos was analyzed by uorescence microscopy (Zeiss Axiozoom V16). Quanti cation of the intensity of rhodamine123 in the intestine area was performed by the software package Fiji [19] Embryotoxicity experiments For determining the toxicities of vinblastine (Signa, V1377) and doxorubicin (Sigma, D1515), 10 embryos were incubated in a 24 well plate with 1 mL test solutions from 6 hpf until 48 dpf to examine developmental abnormalities. A nal abnormality count was performed at 48 hours, and embryos were declared abnormal if at least one of the following criteria applied: i) shortened body length, ii) tail or body curvature. Controls contained DMSO used as a solvent.

Intravenous microinjections of Rhodamine123
Embryos of Tg[ k1:EGFP] and Tg[ k1:EGFP]; abcb4 −/− at 3 dpf were immobilized with tricaine and placed in an agarose injection mold. Next, 1 nl of 2 mg/ml Rhodamine123 (Invitrogen, R302) was injected into the cardinal vein of embryos using a standard zebra sh microinjection apparatus. After 1.5 hours of circulation, the brain area of embryos was imaged using uorescence microscopy (Zeiss Axiozoom V16).
For quanti cation of rhodamine123 intensity in the brain, the green uorescent signal outside of the vasculature of the larval brains was analyzed by Fiji [19]. Each group's measured rhodamine123 intensity values were normalized to the basal level of green uorescent intensity outside of vasculature of noninjected Tg[ k1:EGFP].

Statistics
Statistical analyses were carried out with GraphPad Prism software (GraphPad) utilizing Student t-test or ANOVA. Comparisons between groups were performed with the Bonferroni test. Statistical signi cance was de ned as P < 0.05 Declarations Data availability RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-12901.
Con ict of interest: The authors declare that they have no con icts of interest with the contents of this article.

Figure 1
Generation of zebra sh abcb4 knockout mutant using CRISPER/Cas9 system (a) Schematics of the abcb4 mutant alleles generated using CRISPER/Cas9. The 4 th exon of abcb4 was targeted by gRNA. The sequences of the abcb4 wild-type (WT) and 2 nucleotides deletion mutant allele (abcb4 -/-) were illustrated. Quantitative RT-PCR showed abcb4 transcript reduction in the embryos (b) and brain tissue (c) of the abcb4 mutant. Data are expressed as mean ± SD from at least three independent experiments. Pvalues were calculated using a two-tailed t-test or one-way ANOVA.  Zebra sh Abcb4 protein localizes to blood vessels in the zebra sh brain. Brain tissues of WT (a) and abcb4 knockout (b) adult zebra sh, as a negative control, were stained with anti-Pgp antibody F4 (red) as described in the Materials and Method section. Bar =30 mm. (c) F4 antibody staining of whole adult zebra sh. Bar =3 mm. Positive staining (red) was noted in the forebrain (d), intestine (e), and a subset of renal tubes or collecting ducts in the kidney (f). (g) The F4 positive staining (red) in the brain colocalized with k1:GFP positive cells (green). Fluorescence channels were interrogated individually and merged in.
Nuclei were stained with DAPI (blue). Bar =300 mm for (d, e), 200 mm for (f), and 50 mm for (g). and Tg[ k1:EGFP]; abcb4 -/embryos at 3 dpf and allowed to circulate for 1.5 hours before imaging. (b) Representative images of the dorsal view of the larval brain after rhodamine123 injection showed the level of rhodamine123 accumulation in the brain area. (c) Quanti cation of normalized rhodamine123 intensity in the brain area of Tg[ k1:EGFP] and Tg[ k1:EGFP]; abcb4 -/embryos. Data are expressed as mean ± SD from three independent experiments (black dots). N numbers (gray dots) indicate the total number of embryos across the three independent experiments. P-values were calculated using two-way ANOVA. Age-related transcriptome pro ling in abcb4 knockout zebra sh brain (a) Summary of signi cant DE genes from RNA-seq analysis of brain tissues between WT and abcb4knockout zebra sh at 2 and 30 months. Gene Set Enrichment Analysis (GSEA) of age-associated DE genes (FDR < 0.5) in brain tissue of WT (b) and abcb4 mutant (c) (see Additional le 2 for detailed gene lists of GSEA). (d) Radial graph